Apoptosis and Cell Proliferation, страница 9

Increased backgroundcould occur due to activation of nucleases during sample preparation.DNA Ladder Assay, radioactive17DNA fragmentation(LMW and HMW bysize)g-[32P]-ATP,postlabelP Cellular DNA is isolated by extraction and quickly purified.P Purified total DNA (LMW and HMW DNA) is labeled at the 5’endwith g-[32P]-ATP by T4 Polynucleotide Kinase.P [32P]-labeled DNA is separated by agarose gel electrophoresisand quantitated in the dried gel by a blot analyzer.P Definitive marker of apoptosis: demonstration of the mono- andoligonucleosomal DNA fragments (180 bp multimers)P No prelabeling of the cells required: not limited to cells whichproliferate in vitroP Highly sensitive (1000 x more sensitive than ethidium bromide):allows earlier detection of DNA fragmentation after induction ofapoptosisP Labor-intensive and time-consuming:only a few tests may be performed simultaneouslyP Radioactive assay (32P)P End-labeling of purified DNA requiredProtease Activity AssayActivation ofcaspases(Caspase 3)noneP Apoptotic process including activation of the caspase cascadeis induced in cells by desired method.P Cells are lysed and cell extracts are prepared.P Activated caspase 3 is captured out of cellular lysates by anAnti-caspase 3 antibodyP Quantification of fluorochromes cleaved from a caspasespecific substrate.P Quantitative assay, cleavage of substrate is proportional toconcentration of activated caspase 3 in samplesP Detection of very early stages of apoptosisP Highly specific for caspase 3, no cross reactions with other membersof the caspase familyP High cell numbers neededP Fluorescence reader, equipped with special fluorescence filtersneededpage 17of this guideDiscrete cleavage ofDNA repair enzyme(PARP)noneP Cells are treated with an apoptosis-inducing agent, whichleads to induction of caspase 3 and the cleavage of Poly-ADPRibose-Polymerase (PARP).P Cell extracts are prepared with SDS, fractionated by SDSPAGE, and transferred to a PVDF membrane by westernblotting.P Blot is probed with an antibody to PARP, then with aperoxidase-labeled secondary antibody.P Cleavage products of PARP (about 85 kD) on the membrane arerevealed after an incubation with a peroxidase substrate.P Flexible, can be used with many different types of cellsP No prelabeling of cells required: not limited to cells which proliferatein vitroP Non-radioactiveP Marker for very early stage of apoptosisP Insensitive (requires 105–106 cells/test)P Labor-intensive and time-consuming:only a few tests may be performed simultaneouslypage 20of this guideCellular DNA Fragmentation ELISAApoptotic DNA Ladder KitNucleosome Quantification ELISA13Cell Death Detection ELISAPLUSCaspase 3 Acitivity AssayProtease Activity AssayAnti-PARPG Table 4: Methods for studying apoptosis in cell populations.Sensitive (103–104 cells/test required)Labeled cells do not have to be washedOptimal for microtiter plate formatNon-radioactiveSuitable for analysis of cell-mediated (cytotoxicity) effectsFor productinformation, seeRadioactive isotopeRequires prelabeling and extensive washing of the target cellsLimited to target cells proliferating in vitroIncreased background due to free [3H]-TdR in the cytoplasmP Prelabeling of the target cells requiredP Can only assay target cells proliferating in vitroP Narrow range of quantitative measurement (only one order ofmagnitude)page 54of this guidepage 11of this guide23Cell Death – Apoptosis and NecrosisCell Death – Apoptosis and Necrosis1Apoptosis Assay MethodsApoptosis Assay Methods1Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsMethods for studying apoptosis in individual cells1.2.2 Methods for studying apoptosisin individual cellsIn general, two different labeling methods may be used to identify DNA inapoptotic cells:A number of methods have now been developed to study apoptosis in individualcells.

In the following sections, we will describe details of several of these apoptosisassays.P Enzymatic labeling: Cellular DNAis labeled with modified nucleotides(e.g., biotin-dUTP, DIG-dUTP,fluorescein-dUTP) using exogenousenzymes (e.g., terminal transferase,DNA polymerase). This labeling detects extensive DNA strand breaks.Enzymatic labeling is discussed indetail below (section 1.2.2.1 of thisguide).We focus on two key apoptotic events inthe cell:11DNA fragmentation used to studydeath in cell populations may also beused to study death in individual cells.As described in Section 1.2.1.1, DNAcleavage is a hallmark for apoptosis, andassays which measure prelytic DNAfragmentation are especially attractivefor the determination of apoptotic celldeath.The methods used to assess DNAstrand breaks are based on labeling/staining the cellular DNA.

The labeled/stained DNA is subsequently analyzedby flow cytometry, fluorescence microscopy or light microscopy (Figure 14).ApoptosisHMW DNAenzymaticlabelingvisualizationquantificationapoptotic cellsParameterDNA strand breaksLMW DNApermeabilizationstainingwithfluorochromealtered nucleus morphology2In addition, individual cell death may bestudied by assays that measure alterations in plasma membranes (alterationsin the asymmetry or permeability of individual cell membranes, which occuras the membrane shrinks and becomesincreasingly convoluted.) For instance,during apoptosis, phosphatidylserinetranslocates from the cytoplasmic sideof the membrane to the extracellularside and can be detected with Annexin V(described in section 1.2.2.2 of thisguide).1.2.2.1 The TUNEL enzymatic labelingassaystainingwithfluorochromeReduced DNAcontentG Figure 14: Schematic illustration of the two basic principles for detecting DNA fragmentation insingle cells.24P Staining with fluorochromes: Cellular DNA is stained with fluorescent DNA-binding dyes (DNAfluorochromes) capable of intercalating into DNA.

Upon binding toDNA these dyes become highlyfluorescent. Apoptotic cells are binding less dye molecules, since theycharacteristically lose DNA duringthe staining process (described insection 1.2.2.3 of this guide).Extensive DNA degradation is a characteristic event which often occurs in the earlystages of apoptosis.

Cleavage of the DNAmay yield double-stranded, LMW DNAfragments (mono- and oligonucleosomes)as well as single strand breaks (“nicks”)in HMW-DNA. Those DNA strandbreaks can be detected by enzymatic labeling of the free 3’-OH termini with modified nucleotides (X-dUTP, X = biotin,DIG or fluorescein).

Suitable labelingenzymes include DNA polymerase (nicktranslation) and terminal deoxynucleotidyltransferase (end labeling) (Figure 15).In Situ Nick Translation (ISNT)(template dependent)In Situ End Labeling (TUNEL)(template independent)nick3’5’3’5’+ DNA polymerase+ X-dNTP ( )+ Terminal transferase+ X-dNTP ( )3’5’DNA polymerase I catalyzes the templatedependent addition of nucleotides whenone strand of a double-stranded DNA molecule is nicked.

Theoretically, this reaction(In Situ Nick Translation, ISNT) shoulddetect not only apoptotic DNA, but alsothe random fragmentation of DNA bymultiple endonucleases occurring in cellular necrosis.Terminal deoxynucleotidyl transferase(TdT) is able to label blunt ends of doublestranded DNA breaks independent of atemplate.

The end-labeling method has alsobeen termed TUNEL (TdT-mediated XdUTP nick end labeling)18.The TUNEL method is more sensitive andfaster than the ISNT method. In addition,in early stages cells undergoing apoptosiswere preferentially labeled by the TUNEL13’5’reaction, whereas necrotic cells were identified by ISNT. Thus, experiments suggestthe TUNEL reaction is more specific forapoptosis and the combined use of theTUNEL and nick translation techniquesmay be helpful to differentiate cellularapoptosis and necrosis19.Note: For a comparison of results with theTUNEL and ISNT methods, see Figure 16.To allow exogenous enzymes to enter thecell, the plasma membrane has to be permeabilized prior to the enzymatic reaction.To avoid loss of LMW DNA from the permeabilized cells, the cells have to be fixedwith formaldehyde or glutaraldehyde before permeabilization.

This fixation crosslinks LMW DNA to other cellular constituents and precludes its extraction duringthe permeabilization step.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsThe TUNEL enzymatic labeling assayF Figure 15: Schematic illustration oftwo enzymatic DNA labeling methodsnick translation and end labeling.H Figure 16: Comparison of TUNELand ISNT methods for detectingapoptosis in CD8+ T cells from TcRtransgenic mice. F5 mice are transgenic for a T cell receptor (TcR) specificfor a peptide derived from a nucleoprotein of influenza virus ANT/1968.

In thisexperiment, the nucleopeptide proteinwas injected into F5 mice to activateT cells in vivo. After 4 days, mice weresacrificed and lymphoid organs wereremoved. Cell suspensions were prepared and incubated 4 h at 37°C. Toallow detection of T cells which weredying after the in vivo immune response[Pihlgren, M., Thomas, J. and Marvel, J.(1996) Biochemica, No. 3, 12–14], cellswere stained for CD8 (with a fluorescentantibody), fixed, permeabilized, andthen labeled by either the TUNEL (TdTmediated dUTP Nick End Labeling) orthe ISNT (In Situ Nick Translation) method. Labeled and control cells were analyzed by flow cytometry, with CD8+cells gated.

Spleen cells from a control(not immunized) mouse (red) and fromtwo mice immunized 4 days earlier(green) are shown.Result: The TUNEL method detectedapproximately 15% apoptotic cellsamong CD8+ T cells from the immunized mice. No positive cells werefound in the control mouse. In contrast,the ISNT method was unable to detectany apoptotic cells, possibly due to thelower sensitivity of the technique.25The TUNEL enzymatic labeling assayIf free 3’ ends in DNA are labeled with biotin-dUTP or DIG-dUTP, the incorporatednucleotides may be detected in a second incubation step with (strept)avidin or an antiDIG antibody. The immunocomplex is easily visible if the (strept)avidin or an antiDIG antibody is conjugated with a reporter molecule (e.g., fluorescein, AP, POD).In contrast, the use of fluorescein-dUTP tolabel the DNA strand breaks allows the detection of the incorporated nucleotides directly with a fluorescence microscope ora flow cytometer20.